Search for low-molecular-weight compounds that specifically bind UV-damaged DNA

制备了地塞米松A的靶序列(AATH AATT)上含有(6-4)光产物或环丁烷嘧啶二聚体(CPD)的DNA双链(20个碱基)，并用圆二色谱(CD)分析了地沙霉素的结合。对于含有(6-4)光产物的双链，在长波长区检测到诱导的CD信号，显示地塞米松结合。对于含有CPD的双链，只观察到了很小的诱导。在高盐浓度下使用14-bp双链进行了仔细的滴定实验，以避免非特异性结合，曲线拟合分析表明，含有光产物的DNA的亲和力降低，化学计量比为2.50。与正常靶DNA和损伤DNA形成的络合物的诱导CD差谱分别与文献报道的1：1和2：1络合物的差谱一致。即使在低药物浓度下也能观察到2：1的结合，这一结果表明地塞米松A对(6-4)光产物DNA总是表现出2：1的结合模式。为了比较地沙霉素A与人紫外线损伤的DNA结合蛋白的结合作用，获得了地沙霉素A的识别光谱，该光谱与以前对该蛋白质的识别光谱基本相同。这些结果表明，地塞米松A是一种很好的先导化合物，可以获得与紫外线损伤的DNA特异结合的分子。针对紫外线损伤的DNA人工修复体系的发展，我们分析了(6-4)光产物的碱解反应，发现第一步是5‘组分的N3-C4键的水解。

DNA duplexes (20-bp long) containing the (6-4) photoproduct or the cyclobutane pyrimidine dimer (CPD) at the target sequence for distamycin A, AATh AATT, were prepared, and distamycin binding was analyzed by CD spectroscopy. Induced CD signals at the long wavelength region, which showed the distamycin binding, were detected for the duplex containing the (6-4) photoproduct. Only a small induction was observed for the CPD-containing duplex. Careful titration experiments were carried out using 14-bp duplexes at a high salt concentration to avoid nonspecific binding, and the curve fitting analysis revealed a reduced affinity and a stoichiometry of 2.50 for the photoproduct-containing DNA. The induced CD difference spectra obtained for the complexes with the normal target DNA and the damaged DNA were identical with those reported previously for the 1:1 and 2:1 complexes, respectively. The 2:1 binding was observed even at low drug concentrations, and this result indicated that distamycin A always shows the 2:1 binding mode for the (6-4) photoproduct-containing DNA. In order to compare the binding of distamycin A with that of the human UV-damaged DNA-binding protein, which recognizes DNA containing the (6-4) photoproduct in human cells, the recognition spectrum of distamycin A was obtained, and the spectrum was almost the same as that obtained previously for the protein. From these results, it is concluded that distamycin A is a good lead compound to obtain molecules that specifically bind the UV-damaged DNA. Toward the development of the artificial repair system of the UV-damaged DNA, we analyzed the alkali degradation reaction of the (6-4) photoproduct and found that the first step was hydrolysis of the N3-C4 bond of the 5' component.